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Below you will find an overview of the methods and models used at the Center for Pharmacology. We are happy to support you with our expertise in your research project.

Animal models

Our research uses animal models of peripheral (PNS) and central nervous system (CNS) injuries or heart failure. These models are used to study pathological processes as they occur in humans. Genetically modified mice are also used to analyze the influence of certain genes/proteins specifically. These mouse lines partly represent disease models for diabetes or heart failure, which we use to establish the connection to the phenotype in vivo beyond (patho-) physiological investigations on the cellular and molecular level. We place very high-quality demands on ourselves, animal welfare, and the respective model to achieve the greatest possible significance of the studies. The evaluation is carried out, among other things, using various sensory and motor behavior tests as well as histopathologically, e.g., through immunohistological staining of the tissues. In this way, we can characterize the models both functionally and morphologically and, for example, reliably assess the success of pharmacological interventions.

In vivo neuroprotection

In addition to the injury models listed in the table, which we mainly use in regeneration studies, we also investigate neuroprotective mechanisms. Here, we look at the survival of retinal ganglion cells after optic nerve injury (transection), e.g., by retrograde staining of the neurons. Pharmacologically or using gene therapy, we prevent neuronal death so that regeneration of the severed axons can occur. Substances tested for their neuroprotective properties can be applied systemically or directly into the vitreous (intravitreal) of the eye.

Cell culture

Certain questions can be better addressed in a less complex system. Therefore, we do not only use animal models for our research but initially cell cultures. Besides established cell lines, we mainly use primary cells of the posterior root ganglion or retinal ganglion cells.

Molecular Biology

We have a wide range of molecular biological methods at our disposal to study cellular processes of our model systems, such as gene expression after injury or during regeneration or the influence of pharmacologically active substances on specific signaling pathways. We can also identify and characterize proteins that may be potential therapeutic targets. The expression of recombinant proteins or the production of viral vectors for gene therapy approaches is also performed.

Pharmacology

Using pharmacological methods, we aim to improve the regeneration of the peripheral and central nervous system after injury and thus contribute to developing new drugs.

In animal models, we can investigate different substances' efficacy, dosage, and optimal route of administration (e.g., intraperitoneal, oral, or intravenous). Functional behavioral tests and histological and anatomical examinations provide us with information about the efficacy of the substances (mode of action) and potential clinical applications if proof of effectiveness is achieved (proof of concept). Using molecular biology methods and cell culture, we can decipher the underlying mechanism of action (Mode of Action).

Gene therapy

We are interested in the possibility of pharmacologically influencing the regeneration of nerve tissue and gene therapy. In this process, nucleic acid (DNA or RNA) is introduced into the cells of an individual, e.g., to exchange a defective gene, which is causative for a disease, for a healthy one, or simply to express it. The expression of essential signal molecules can also be controlled in this way, and the course of the disease can be positively influenced. Viruses primarily transfer nucleic acid. However, body cells can be removed, genetically modified, and re-implanted. Gene therapy medicinal products belong to the group of advanced therapy medicinal products. So far,  few gene therapeutics are on the market because the technology is still relatively young, and regulatory authorities have strict quality requirements for clinical trials to ensure patient safety.

Isolation of cardiac muscle cells

For our patch clamp experiments, we use, among others, isolated mouse cardiac myocytes (ventricular myocytes). We isolate these cells fresh, as they are difficult to culture and rapidly lose their typical properties or are no longer suitable for all studies. Ventricular myocytes are special by their morphology alone because they are elongated and relatively large, and their cell membrane has invaginations (so-called T-tubules), which are essential for electromechanical coupling ("excitation-contraction coupling"). In addition to morphology, protein expression and localization cannot be mimicked in cultured cells such as those of the widely used HEK293 line, which we also use, so many studies on the (patho-) physiological regulation of, e.g., ion channels have to be performed on primary cells. For this purpose, we use, among others, ventricular myocytes of genetically modified mice [please link to the section “Animal Models”].

Patch clamp method

For many years we have been performing electrophysiological studies on cells from cell culture (mainly HEK293) or on freshly isolated primary cells, mainly mouse ventricular myocytes. The patch clamp technique is used. In the “whole-cell configuration,” we measure currents that flow across the entire cell membrane, as would be the case, for example, with an action potential. In the “cell-attached configuration," we can measure currents flowing through a single ion channel. So here we can watch a single molecule "at work" live.

Pharmacology

We use drugs very predominantly as "tools" to selectively modulate the function of certain molecules. In patch-clamp studies, for example, we can measure ion currents flowing through a particular type of ion channel by combining them with the appropriate measurement protocol (including holding potential, test potential, frequency, and duration of a potential change). On the one hand, we can selectively suppress currents that are not of interest, or on the other hand, we can induce a change in the current response that is specific for a certain type of ion channel, e.g., to identify it. In part, however, we are also investigating new drugs with the question of whether and what influence they have on, for example, ion channels or modulating G proteins.

Pharmacokinetic biomarkers

The kinetics of drugs (pharmacokinetics) and other xenobiotics is determined by the activity of underlying pharmacokinetic processes (including glomerular filtration, turnover by xenobiotic-metabolizing enzymes, and transporters). Based on in vitro data, the prediction of the activities of these processes in humans is limited. Therefore, we develop and validate, among others using clinical studies, methods to selectively measure such activities in individual subjects or patients using endogenous or exogenous marker substances. Pharmacokinetic biomarkers also enable the quantification of corresponding influencing factors (e.g., in drug interactions) and contribute to an individually optimized dosage of drugs.

Clinical studies in healthy volunteers

Clinical studies in healthy volunteers are indispensable for determining the safety and tolerability, pharmacokinetics, and effects of drugs in humans. We conduct clinical studies on our volunteer ward (12 beds) by the German Drug Law (AMG) and outside the regulations of the AMG. Quality is ensured by adherence to the applicable GCP (good clinical practice) guidelines and by following our standard operating procedures (SOPs). We can carry out all tasks independently, from conception to preparation of the necessary documents, submission to ethics committees and authorities, operational implementation, evaluation, and reporting. Still, we are also happy to work with cooperation partners.

Clinical studies on patients

In cooperation with the clinics of the University Hospital as well as with other cooperation partners, we participate in clinical studies on patients in which the pharmacokinetic question is to be tested. The type of cooperation ranges from limited collaboration about a specific task to leading research, depending on the research question and the preferences of the cooperation partners.

Population pharmacokinetic/pharmacodynamic modeling and simulation

Using this "top-down" method (software: NONMEM, Monolix, among others), we determine the pharmacokinetic and pharmacodynamic properties of drugs and other xenobiotics from experimental data of clinical studies and simultaneously record the variability of drug concentrations in the population under investigation. The contribution of influencing factors such as the activity of underlying pharmacokinetic processes (see section "Pharmacokinetic Biomarkers") or anthropometric data to the overall variability can be tested and quantified.

Physiology-based pharmacokinetic (PBPK) modeling of drugs

With PBPK modeling of drugs (software: e.g., PK-SIM) we try to describe and predict the concentration-time courses of drugs in the organs and tissues of an organism based on the physicochemical properties of a substance and the activity of pharmacokinetic processes relevant to this substance ("bottom-up" method). Suppose the predictions of such models are in good agreement with experimentally collected data. In that case, they can also make predictions and provide dose recommendations for unstudied populations (e.g., the elderly) without the need to conduct new clinical studies for each question.

Determination of drug concentrations with high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS).

The sensitive, specific, and precise measurement of drug concentrations in biological fluids, buffers, and media is an essential prerequisite for determining their behavior in any system (in vitro, animal studies, clinical trials). We can use several triple quadrupole mass spectrometers to quantify such concentrations, sometimes down to the pg/ml range. This technique, widely available for almost two decades, is adequate for dealing with our questions.

Determination of human cytochrome P450 (CYP) enzyme activity in vitro

CYPs are often the rate-determining enzymes in human drug metabolism. Reliable methods for activity determination in vitro, which also allow high throughput, are essential for the initial characterization of drugs as substrates or inhibitors of the major CYPs. By combining different substrates in an "in vitro - cocktail" and using advanced evaluation methods, the nature and extent of interaction of the drugs with the enzymes can be reliably determined.

Preparation of target proteins

For the identification and initial investigation of new drug candidates (ligands), it is necessary to have the corresponding target structures (targets) available in purified form. Most of such targets are recombinant proteins, which we produce using prokaryotic or eukaryotic overexpression systems and purify by various column chromatographic methods. Eukaryotic cells are used here whenever the target protein contains post-translational modifications of which bacterial cells are incapable. The proteins obtained in this way are first examined concerning their purity and identity before being used in various activity or binding assays for the identification/characterization of ligands. In addition, such proteins are provided to collaborators for studying target-ligand complexes by X-ray crystallography.

Development of high-throughput assay methods

To qualitatively and quantitatively characterize the direct interaction of a ligand with its target, biochemical test methods (assays) are required that detect the influence of the binding of two molecules (binding assay) or the modulation of the activity of enzymes (activity assay). We use spectrophotometric or fluorescence-based methods and microscale thermophoresis for many of these assays. Accordingly, the first step is to develop chromogenic or fluorogenic enzyme substrates or to generate fluorescently labeled probes with high binding affinity for the target proteins and characterize them on the targets. To validate newly developed assays for the search of drug candidates, we first investigate literature-known ligands concerning their behavior towards the system of target and substrate(s)/probe(s) and determine the dissociation constant of the literature-known ligands. Furthermore, we optimize the assays for investigating substance libraries in a high-throughput procedure using 96- or 384-well microtiter plates. Finally, the validated and optimized assays are used to identify and characterize new ligands.

Identification and characterization of ligands

For the identification and the initial investigation of new drug candidates (ligands), we first look at the interaction of a large number of compounds with the target. For this purpose, we perform so-called high-throughput screening campaigns of compound libraries on recombinant target proteins previously generated in prokaryotic or eukaryotic overexpression systems, using binding or activity assays developed for this purpose. Identified "hits" are further quantitatively characterized concerning their interaction with the target to derive structure-activity relationships, which are the starting point for further optimization of the ligands concerning their binding affinity. In addition, selected ligands are co-crystallized with the target, providing information on the precise binding behavior of the ligands.

Determination of human cytochrome P450 (CYP) enzyme activity in vitro

CYPs are often the rate-determining enzymes in human drug metabolism. Reliable methods for activity determination in vitro, which also allow high throughput, are essential for the initial characterization of drugs as substrates or inhibitors of the major CYPs. By combining different substrates in an "in vitro - cocktail" and using advanced evaluation methods, the nature and extent of interaction of the drugs with the enzymes can be reliably determined.